There is a complex, conserved molecular chaperon machinery in all cells that mediates folding and assembly of proteins in general. Normal interactions of proteins with[unreadable] eh chaperon machinery are short-lived, but a number of key regulatory proteins have extended interactions with certain chaperon components; in a poorly defined manner, these extended interactions appear to serve a regulatory purpose. The broad, long-term objectives of this proposal are to gain a better understanding of how molecular chaperones are used in controlling the function of diverse regulators of cell growth and differentiation and how drugs that inhibit chaperon activity may be used to treat conditions resulting from the actions of particular regulatory proteins. The present proposal is designed to test the hypothesis that chaperoned regulation is a common regulatory mechanism that participates in directing diverse signal transduction pathways. Steroid receptors are perhaps the best characterized regulatory proteins displaying extended interactions with the chaperon machinery. A cell-free system based on rabbit reticulocyte lysate has been shown to faithfully reconstitute progesterone receptor (PR) complexes in vitro, and this system has been used to identify multiple chaperon components involved ina dynamic assembly process. The following Specific Aims are intended to better characterize athe chaperon components involved and too demonstrate common chaperon interactions with regulatory proteins unrelated to steroid receptors: 1. Extend studies on p60, a key chaperon component in assembly of PR complexes 2. Examine the role of a novel protein, p48, in assembly of PR complexes 3. Examine the ability of geldanamycin A to inhibit hsp90 interactions with PR and characterize the nature of hsp90 interactions with t his drug 4. Extend studies on chaperon interactions to regulatory proteins other than PR The research design calls for 1) development of immunological and molecular reagents to study chaperones found in the rabbit reticulocyte lysate cell-free assembly system and 2) the addition of potential targets for chaperoned regulation to the cell-free system. Achieving the aims will advance knowledge of the chaperon machinery and its role in directing the activities of key regulators of cellular growth and differentiation.